© 2014. Published by The Company of Biologists Ltd | Development (2014) 141, 1961-1970 doi:10.1242/dev.106310
RESEARCH ARTICLE
Noonan and LEOPARD syndrome Shp2 variants induce heart
displacement defects in zebrafish
ABSTRACT
Germline mutations in PTPN11, encoding Shp2, cause Noonan
syndrome (NS) and LEOPARD syndrome (LS), two developmental
disorders that are characterized by multiple overlapping symptoms.
Interestingly, Shp2 catalytic activity is enhanced by NS mutations and
reduced by LS mutations. Defective cardiac development is a
prominent symptom of both NS and LS, but how the Shp2 variants
affect cardiac development is unclear. Here, we have expressed the
most common NS and LS Shp2-variants in zebrafish embryos to
investigate their role in cardiac development in vivo. Heart function
was impaired in embryos expressing NS and LS variants of Shp2.
The cardiac anomalies first occurred during elongation of the heart
tube and consisted of reduced cardiomyocyte migration, coupled with
impaired leftward heart displacement. Expression of specific laterality
markers was randomized in embryos expressing NS and LS variants
of Shp2. Ciliogenesis and cilia function in Kupffer’s vesicle was
impaired, likely accounting for the left/right asymmetry defects.
Mitogen-activated protein kinase (MAPK) signaling was activated to
a similar extent in embryos expressing NS and LS Shp2 variants.
Interestingly, inhibition of MAPK signaling prior to gastrulation
rescued cilia length and heart laterality defects. These results
suggest that NS and LS Shp2 variant-mediated hyperactivation of
MAPK signaling leads to impaired cilia function in Kupffer’s vesicle,
causing left-right asymmetry defects and defective early cardiac
development.
KEY WORDS: Noonan syndrome, LEOPARD syndrome, Shp2,
MAPK, Zebrafish
INTRODUCTION
PTPN11 encodes Shp2 (Ptpn11 – Zebrafish Information Network),
a ubiquitously expressed non-receptor protein-tyrosine phosphatase
(PTP) with two Src homology 2 (SH2) domains (Freeman et al.,
1992; Feng et al., 1993; Chan and Feng, 2007) that is involved in a
variety of signal transduction processes, such as the Ras-Raf-MAP
kinase (Tidyman and Rauen, 2009), Jak-Stat (Freeman et al., 1992;
Neel et al., 2003) and phosphatidylinositol-3 kinase (PI-3K)
pathways (Feng, 1999). Shp2 plays a crucial role in the
transduction of the signal from receptor tyrosine kinases (RTKs),
including the receptors of platelet-derived growth factor (Pdgfr)
(Van Vactor et al., 1998), fibroblast growth factor (Fgfr) (Neel and
1
Hubrecht Institute-KNAW and University Medical Center Utrecht, Utrecht 3584 CT,
2
The Netherlands. Institute of Biology, Leiden 2333 CC, The Netherlands.
*Author for correspondence ( [email protected])
This is an Open Access article distributed under the terms of the Creative Commons Attribution
License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,
distribution and reproduction in any medium provided that the original work is properly attributed.
Received 21 November 2013; Accepted 11 March 2014
Tonks, 1997) and the epidermal growth factor (Egfr) (Huyer and
Alexander, 1999; Qu, 2000), as well as from cytokine receptors
and integrins (Neel and Tonks, 1997; Huyer and Alexander, 1999).
Missense germline mutations in PTPN11 are associated with
Noonan syndrome (NS) (OMIM: 163950) and LEOPARD syndrome
(LS) (OMIM: 151100), two autosomal dominant disorders. NS is a
relatively common disorder that affects 1 in 1000-2000 live births.
Individuals with NS are characterized by congenital heart defects,
including atrial and ventricular septal defects, pulmonary stenosis and
hypertrophic cardiomyopathy (HCM) (Tartaglia et al., 2001; Digilio
et al., 2002). In addition, these individuals display short stature and
facial abnormalities. LS is a more rare disorder (1:3500 live births) that
shows a substantial overlap with the various symptoms of NS with two
more clinical manifestations that are specific for LS: deafness and
‘café au lait’ spots on the skin (Mendez and Opitz, 1985; Legius et al.,
2002). The congenital heart defects among individuals with PTPN11
mutations differ between NS and LS, in that pulmonary stenosis is
most common in NS, whereas HCM prevails in LS (Allanson and
Roberts, 1993; Sarkozy et al., 2008).
Interestingly, the biochemical properties of NS-Shp2 and LSShp2 are distinct, in that NS mutations enhance catalytic activity and
LS mutations strongly reduce PTP activity (Keilhack et al., 2005;
Kontaridis et al., 2006). The crystal structure of Shp2 shows that,
in the absence of a binding partner, the N-SH2 domain interacts with
the PTP domain and blocks the catalytic site (Hof et al., 1998). NS
mutations predominantly reside in the interface between the N-SH2
domain and the PTP domain, resulting in the disruption of the closed
conformation and enhanced catalytic activity of NS-Shp2 (Keilhack
et al., 2005; Nakamura et al., 2009). By contrast, most LS mutations
reside close to the active site and result in strongly reduced, yet
detectable, catalytic activity (Keilhack et al., 2005; Hanna et al.,
2006; Yu et al., 2013). How two mutations with opposite effects on
catalytic activity result in syndromes withsimilar clinical symptoms
is a conundrum that still needs to be resolved.
NS-Shp2 causes upregulation of the MAPK pathway (Feng, 1999).
Whereas several studies show that LS mutations downregulate the
level of phosphorylated ERK (Kontaridis et al., 2006; Stewart et al.,
2010), the role of LS-Shp2 mutation on the MAPK pathway is still
controversial (Oishi et al., 2006, 2009; Edouard et al., 2010).
Increased RAS/MAPK signaling is implicated in the gain-of-function
phenotypes that are caused by expression of the LS Drosophila
ortholog of PTPN11, corkscrew (csw) (Oishi et al., 2009), suggesting
a mechanism by which NS and LS variants may result in similar
phenotypes. However, NS and LS signaling are distinct, because it is
well established that LS mutations, but not NS mutations (De Rocca
Serra-Nedelec et al., 2012), enhance PI3K/AKT signaling in hearts of
LS/+ knock-in mice (Kontaridis et al., 2006) as well as in fibroblasts
isolated from individuals with LS (Edouard et al., 2010). The
similarities in RAS/MAPK signaling of NS and LS variants may
underlie the overlapping symptoms in NS and LS, whereas the
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DEVELOPMENT
Monica Bonetti1, Jeroen Paardekooper Overman1, Federico Tessadori1, Emily Noë l1, Jeroen Bakkers1 and
Jeroen den Hertog1,2,*
differences in PI3K/Akt signaling may cause the differences in
NS- and LS-induced developmental defects.
Shp2 knockout, knockdown and gain-of-function studies in a
variety of organisms have begun to reveal the roles of Shp2 in
embryonic development. Shp2-null mouse embryos die preimplantation due to defective Erk activation and trophoblast stem
cell death (Yang et al., 2006). Dominant-negative Shp2 mutants
disrupt gastrulation in Xenopus (Tang et al., 1995). Moreover,
mouse models have been generated for the two most prevalent
NS and LS mutations, Ptpn11-D61G/+ and Ptpn11-Y279C/+,
respectively. These mice exhibit developmental defects, including
reduced length, craniofacial abnormalities and congenital heart
defects, reminiscent of the clinical characteristics of the human
disorders (Araki et al., 2004; Marin et al., 2011). In particular,
pulmonary stenosis and HCM are evident in the NS and LS mice,
respectively. Shp2 is conserved in zebrafish and we previously
generated the most common human NS and LS mutations in a
cDNA encoding full-length zebrafish Shp2. Micro-injection of
mRNA-encoding mutant Shp2 variants into zebrafish embryos at
the one-cell stage induces gastrulation cell movement defects, as
well as craniofacial and cardiac defects (Jopling et al., 2007).
Furthermore, Stewart et al. used zebrafish embryos to show that the
function of Shp2 in neural crest cells underlies LS pathogenesis
(Stewart et al., 2010).
The role of NS and LS Shp2 variants in the development of
cardiac defects is still unclear. Zebrafish has become a powerful
model with which to study cardiac development in recent years
(Bakkers, 2011; Lien et al., 2012). We set out to study early heart
development in zebrafish embryos expressing NS and LS variants
of Shp2, taking advantage of the transparency of zebrafish
embryos, which facilitates time-lapse analysis of the onset and
nature of the cardiac defects in vivo. Our analyses revealed
impaired heart function and morphogenesis, which were highly
similar in NS- and LS-Shp2-expressing embryos. Furthermore, the
heart defects were accompanied by randomization of left-right
asymmetry, which was probably due to impaired ciliogenesis and
defective cilia function in Kupffer’s vesicle that we observed in
early embryos. Treatment with a MEK-inhibitor, CI-1040, prior to
gastrulation rescued defective leftward heart displacement and the
laterality defects in NS- and LS-Shp2-expressing embryos. Taken
together, our results provide insight into the mechanism by which
NS and LS Shp2 variants induced laterality defects through
hyperactivation of MAPK signaling, resulting in cardiac defects
during early development.
RESULTS
Expression of NS and LS Shp2 variants caused defects in
cardiac function
To investigate the role of Shp2 variants in cardiac function, Shp2D61G and Shp2-A462T mRNAs were injected at the one-cell stage.
In order to monitor expression of mutant Shp2, we fused a green
fluorescent protein (GFP)-peptide 2A sequence to the N terminus of
Shp2, which is cleaved off auto-proteolytically (Kim et al., 2011)
(supplementary material Fig. S1). Following expression of (mutant)
Shp2, the developing heart was analyzed at 55 hpf by high-speed
video recording. Kymographs of the atrium and ventricle were
generated, allowing quantification of heart function, as described
previously (Tessadori et al., 2012). Representative examples of
kymographs of embryos expressing WT-Shp2 and Shp2-D61G are
depicted in Fig. 1A-F. Quantification of the kymographs of Shp2WT-, Shp2-D61G- and Shp2-A462T-injected embryos revealed
significantly longer cardiac cycles in the Shp2 variant-expressing
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Development (2014) 141, 1961-1970 doi:10.1242/dev.106310
Fig. 1. Impaired cardiac function in embryos expressing Shp2-D61G
and Shp2-A462T. (A,B) The hearts of WT-Shp2- (Shp2) and Shp2-D61Gexpressing zebrafish embryos were imaged by high-speed video recording
microscopy at 2 dpf. White dotted lines through the atrium (a) and the
ventricle (v) are indicated. (C-F) Ventricular (C,D) and atrial (E,F)
kymographs from 2 dpf embryonic hearts. Note the longer period of
the Shp2-D61G-expressing heart, compared with the WT-Shp2-expressing
heart (double-headed arrow and white dotted vertical lines). (G) Quantitative
analysis of the heart period in the ventricle or atrium of WT-Shp2, Shp2-D61G
and Shp2-A462T expressing embryos (n indicates number). Whisker plots
are depicted.
embryos, reflecting a reduced but regular heart rate (Fig. 1G). Other
functional defects, such as an atrioventricular block or uncoupling
of the two chambers, were not detected in Shp2-D61G- and Shp2A462T-expressing embryos. These results illustrate functional
cardiac defects, particularly a reduced heart rate, in embryos
expressing NS and LS variants of Shp2.
Impaired heart asymmetry in embryos expressing NS and LS
Shp2 variants
To further characterize the cardiac phenotype in response to
expressing of NS and LS variants of Shp2, we first examined the
overall morphology and regionalization of the heart at 55 hpf by in situ
hybridization (ISH), using a panel of cardiac markers. At this stage, the
heart chambers are completely formed in wild-type embryos and
the heart undergoes looping morphogenesis. Analysis of heart shape
using a myl7-specific probe (formerly known as cmlc2) revealed that
the hearts of Shp2-D61G- and Shp2-A462T-expressing embryos
exhibited either inverted looping (14% and 11%, respectively) or nonlooped heart (39% and 25%, respectively) in contrast to non-injected
DEVELOPMENT
RESEARCH ARTICLE
Fig. 2. Heart defects in embryos expressing Shp2-D61G and
Shp2-A462T. Non-injected control embryos (NIC) and embryos injected at
the one-cell stage with mRNA encoding WT-Shp2 (Shp2), Shp2-D61G or
Shp2-A462T were fixed at 48 hpf and in situ hybridization was performed
using probes for the myosin genes myl7 (A-D, cardiomyocytes), vmhc
(E-H, ventricle) and amhc (I-L, atrium), and for has2 (M-P, endocardial
cushions). Representative pictures are shown and the number of embryos
showing this pattern/total number of embryos is indicated. The outline of the
heart is indicated in M-P.
control and WT-Shp2-injected embryos (Fig. 2A-D). Myocardial
chamber specification was not affected in NS-Shp2- and LS-Shp2injected embryos, as determined by expression of ventricular myosin
heavy chain (vmhc) (Fig. 2E-H) and atrium myosin heavy chain
(amhc; myh6 – Zebrafish Information Network) (Fig. 2I-L). Next, the
Development (2014) 141, 1961-1970 doi:10.1242/dev.106310
formation of the endocardial cushions, from which the atrioventriclar
valves will develop, was assessed by has2 expression. In Shp2-D61Gand Shp2-A462T-expressing embryos, has2 expression was not
affected, compared with non-injected control and WT-Shp2expressing embryos (Fig. 2M-P). Expression of this panel of
markers was also assessed in embryos expressing Shp2-T73I (NS)
and Shp2-G465A (LS) with similar results (supplementary material
Fig. S2), indicating that all NS and LS variants induced similar
defects. Taken together, these data indicate that expression of NS and
LS variants of Shp2 led to a failure to undergo heart looping in a large
proportion of the embryos, without inducing defects in cardiac
chamber specification or endocardial cushion formation. In addition,
the NS and LS variants of Shp2 induced cardiac defects to a similar
extent during early development.
To investigate at which stage the cardiac defects arise in embryos
expressing NS and LS variants of Shp2, myl7 expression was
analyzed at different time points of heart development, particularly
during cardiac fusion and heart tube elongation. Zebrafish heart
formation is initiated by fusion of two bilateral pools of
cardiomyocytes at the 16-somite stage (16 hpf ) into a cardiac disc
at the midline of the embryo at 22 somites (20 hpf ) (Yelon et al.,
1999; Bakkers, 2011). No obvious defects were detected in
myl7 expression or cardiac fusion between non-injected control,
WT-Shp2-, Shp2-D61G- and Shp2-A462T-expressing embryos up
to 22 somites (Fig. 3A-H). At 28 hpf, the heart in WT-Shp2expressing embryos formed a tube that extended from the midline to
the area under the left eye (Fig. 3I), which resembled normal heart
displacement. By contrast, in Shp2-D61G- and Shp2-A462Texpressing embryos, the displacement of the heart is randomized:
23% of Shp2-D61G-expressing embryos and 28% of Shp2-A462Texpressing embryos had a heart on the right, whereas 34% of
Shp2-D61G-expressing embryos and 24% of Shp2-A462Texpressing embryos had their heart tube at the midline (Fig. 3J-L).
Taken together, these results indicate that the first signs of cardiac
abnormality in embryos expressing Shp2 variants are observed
during asymmetric displacement of the heart tube.
Fig. 3. Randomized heart displacement in response
to expression of Shp2-D61G and Shp2-A462T.
Embryos were injected at the one-cell stage with mRNA
encoding WT-Shp2, Shp2-D61G or Shp2-A462T and
fixed at the 16-somite stage, the 22-somite stage or at
28 hpf. In situ hybridization was carried out using a
myl7-specific probe to mark cardiomyocytes. (A-H) The
number of embryos displaying the depicted pattern/total
number of embryos is indicated. (I-K) Representative
pictures are shown displaying heart displacement to the
left (I), middle (J) and right (K). (L) Percentages of left,
middle and right cardiac displacement are depicted.
The number of embryos (n) is indicated.
DEVELOPMENT
RESEARCH ARTICLE
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Development (2014) 141, 1961-1970 doi:10.1242/dev.106310
Decreased cardiomyocyte migration speed and rotation in
embryos expressing NS and LS Shp2 variants
NS and LS Shp2 variants disrupt left/right asymmetry
To determine whether the loss of asymmetry in the hearts of embryos
expressing NS and LS Shp2 variants is associated with disruption of
overall L/R asymmetry, we analyzed the expression of a number of
laterality markers. southpaw (spaw) is one of the earliest markers
displaying asymmetric expression in development (Long et al.,
2003). spaw was predominantly expressed in the left lateral plate
mesoderm (LPM) in WT-Shp2-expressing embryos (Fig. 5A). spaw
expression was affected in embryos expressing Shp2-D61G and
Shp2-A462T, compared with WT-Shp2, in that 23% of Shp2-D61Gand 17% of Shp2-A462T-expressing embryos displayed bilateral
spaw expression and 26% of Shp2-D61G- and 28% of Shp2-A462Texpressing embryos expressed spaw exclusively in the right LPM
(Fig. 5B-D). Next, expression of lefty2 (lft2), a downstream target of
spaw, was investigated at the 22-somite stage (20 hpf ). In WT-Shp2expressing embryos, lft2 was detected in the left cardiac field in all
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Fig. 4. Impaired cardiomyocyte migration in embryos expressing
Shp2-D61G and Shp2-A462T. Embryos from the tg(myl7:GFP) line were
injected with mRNA encoding WT-Shp2, Shp2-D61G or Shp2-A462T
lacking eGFP-peptide 2A sequences to allow imaging of GFP-positive
cardiomyocytes from the 22-somite stage onwards. (A-F) Representative
individual GFP-positive cells were color-coded according to their location
within the cardiac field at the 22-somite stage and tracked over a 200 min
period. Dorsal view with anterior towards the top. Schematic representations
of the embryos on the left indicate the position and shape of the heart in
green. (A-C) WT-Shp2; (D-F) Shp2-A462T. (G) Quantification of the total
speed of posterior (P) and anterior (A) cardiac progenitor cells. WT-Shp2expressing embryos displayed normal leftward heart displacement; embryos
expressing Shp2-D61G and Shp2-A462T that did not display normal
heart displacement were selected for further analysis. (H) Clockwise
cardiomyocyte rotation was determined of the same embryos as in G. Rotation
was quantified as depicted in I. (G,H) Averages are indicated and error bars
indicate s.e.m.; n indicates number of embryos. Statistical significance was
determined using Student’s t-test: **P<0.01 and ***P<0.001.
cases (Fig. 5E). By contrast, expression of lft2 in Shp2-D61G- and
Shp2-A462T-expressing embryos was randomized. 30% of the
Shp2-D61G-expressing embryos expressed lft2 on the right side
(Fig. 5E-G). Similarly, in Shp2-A462T-expressing embryos, lft2
expression was detected at the right side in 42% of the embryos.
spaw is required for visceral organ L/R asymmetry (Long et al.,
2003), suggesting that defects in L/R asymmetry may not be limited
to the heart in embryos expressing Shp2 variants. To investigate
this, the position of the liver was assessed by analysis of the
expression of fabp. fabp expression was randomized at 48 hpf in
embryos expressing Shp2-D61G and Shp2-A462T, in that the liver
developed on the opposite side in 20% of the Shp2-D61Gexpressing embryos and 24% of the Shp2-A462T-expressing
embryos, or on both sides in 18% of the Shp2-D61G- and 9% of
the Shp2-A462T-expressing embryos (Fig. 5H-K). In control WTShp2-expressing embryos, the liver always developed on the left
side of the embryos. Analysis of the embryonic midline using the
DEVELOPMENT
Randomization of heart displacement may be caused by compromised
cardiomyocyte migration (Ramsdell, 2005). Tg(myl7:GFP) embryos
express eGFP in cardiomyocytes and were used for high-resolution
confocal time-lapse imaging to assess cardiomyocyte migration.
As WT-Shp2-expressing embryos were indistinguishable from
non-injected control embryos in in situ hybridization experiments
(Figs 2 and 3), we used WT-Shp2-expressing embryos as a control for
the embryos expressing NS and LS variants of Shp2 for subsequent
experiments. Single cells were tracked in WT-Shp2-, Shp2-D61G- and
Shp2-A462T-expressing embryos from cardiac disc stage (21 somites,
19.5 hpf) for 4 h and their velocities and directions were determined.
Consistent with observations of heart morphogenesis in wild-type
embryos (de Campos-Baptista et al., 2008; Smith et al., 2008), cardiac
cells moved towards the left and anterior part of the embryo in
WT-Shp2-expressing embryos (n=4). Cardiomyocytes in the posterior
part of the cardiac disc moved faster than anterior cells (Fig. 4A-C).
The Shp2-D61G- and Shp2-A462T-expressing embryos were
imaged at 19.5 hpf, like the WT-Shp2-expressing embryos, and
heart displacement was assessed subsequently at 24 hpf, allowing
correlation between early migration defects and heart displacement.
Embryos that displayed impaired heart displacement were selected for
further analysis. It is noteworthy that none of the selected embryos
displayed heart displacement to the right (Shp2-D61G, n=8; Shp2A462T, n=8), which may be due to low penetrance of rightward heart
displacement. Although cardiomyocytes in the selected embryos
formed a cardiac tube, they showed reduced migration on the left.
Representative images of an embryo expressing Shp2-A462T are
depicted in Fig. 4D-F. Quantification of the speed of cardiomyocytes
confirmed slower migration of cells in embryos expressing Shp2D61G and Shp2-A462T, compared with WT-Shp2 (Fig. 4G).
Cardiomyocytes in selected embryos expressing Shp2-D61G and
Shp2-A462T did not cross the midline, but instead migrated
anteriorly, which is consistent with defective heart displacement to
the left. Rotation of the heart tube was quantified as described before
and the observed 30° clockwise rotation of the cardiac cone in WTShp2-expressing embryos was consistent with the previously reported
clockwise rotation in control embryos (Smith et al., 2008). Clockwise
rotation was significantly decreased in embryos expressing Shp2D61G and Shp2-A462T with impaired leftward heart displacement,
compared with control WT-Shp2 (Fig. 4H). Our results indicate that
directional cardiomyocyte migration and heart tube rotation is reduced
in embryos expressing NS and LS Shp2 variants, which is consistent
with the observed defects in heart tube displacement.
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Development (2014) 141, 1961-1970 doi:10.1242/dev.106310
Fig. 5. Expression of Shp2-D61G- and Shp2A462T-induced L/R asymmetry defects.
Embryos were injected at the one-cell stage with
mRNA encoding WT-Shp2 (Shp2), Shp2-D61G
or Shp2-A462T and fixed at the stage indicated.
In situ hybridization was carried out using
probes specific for spaw (A-D, southpaw), lft2
(E-G, lefty2) and fabp (H-K, fatty acid binding
protein, marking the liver). Representative
pictures are shown of Shp2-D61G-expressing
embryos. lft2 expression was observed only on
the left side or on the right side. Organ
asymmetry as assessed using the different
markers was scored for embryos injected with
WT-Shp2, Shp2-D61G and Shp2-A462T.
Percentages of left, middle/bilateral and right
expression of the markers are depicted. The
number of embryos (n) is indicated.
flow. In addition, beads tracked in Shp2-D61G and Shp2-A462Texpressing embryos showed a strongly reduced average flow velocity
relative to wild-type Shp2-expressing embryos (Fig. 6M). These
results indicate that ciliogenesis and cilia function is impaired in the
KV of embryos expressing NS and LS variants of Shp2, resulting in
defective KV function.
Ciliogenesis and cilia function in Kupffer’s vesicle are
impaired in embryos expressing NS and LS Shp2-variants
Early treatment with the MEK inhibitor CI-1040 rescued L/R
asymmetry, heart asymmetry and KV function in embryos
expressing NS and LS Shp2 variants
It is well established that Kupffer’s vesicle (KV), a fluid-filled
ciliated organ, is involved in the establishment of L/R asymmetry in
zebrafish (Neugebauer et al., 2009; Liu et al., 2011). Through the
oriented rotation of the cilia inside the KV, asymmetric expression of
genes such as spaw and lft2 is established. Immunohistochemistry
was carried out using an acetylated tubulin antibody to detect cilia in
the KV of 10-somite stage embryos (14 hpf ) (Fig. 6A-C). The mean
number of cilia was 51.4±0.5 in WT-Shp2-expressing embryos,
whereas in embryos expressing Shp2-D61G and Shp2-A462T, the
number of cilia was significantly reduced to 30.4±0.7 and 28.5±0.4,
respectively (n=10 embryos each) (Fig. 6D). In addition, the cilia
length was significantly reduced in embryos expressing NS and LS
Shp2 variants (both 3.2±0.1 μm, n=241 cilia for Shp2-D61G and
n=172 cilia for Shp2-A462T, respectively) compared with control
(4.2±0.1 μm, n=379 cilia for WT-Shp2) (Fig. 6E). Measurements of
the area of KV indicated significant differences in lumen size among
wild-type (4517±355 μm2, n=10), Shp2-D61G (2243±190 μm2,
n=10) and Shp2-A462T (1914±256 μm2, n=10) embryos (Fig. 6F).
These results suggest that disruption of L/R asymmetry in embryos
expressing NS and LS variants of Shp2 was a consequence of
defective KV function, resulting from impaired ciliogenesis in the
KV. To determine the KV functionality directly, we analyzed
the fluid flow in embryos expressing NS and LS variants of Shp2.
Tracking of fluorescent beads injected into the KV lumen of wildtype Shp2-expressing embryos showed counterclockwise rotation of
the beads (Fig. 6G). By contrast, tracking of the beads in Shp2D61G- and Shp2-A462T-expressing embryos showed that the beads
moved about randomly (Fig. 6H-I), indicating a loss of coordinated
Shp2 variants enhance RAS/MAPK signaling (Feng, 1999).
Previously, we and others showed that inhibition of MEK rescued
developmental defects in zebrafish embryos that were caused by
expression of activators of the RAS/MAPK pathway (Anastasaki
et al., 2009; Runtuwene et al., 2011). We investigated whether
inhibition of MEK would also rescue the cardiac defects in embryos
expressing NS and LS Shp2 variants. First, the efficacy of the MEK
inhibitor CI-1040 to rescue early developmental defects was
assessed. Expression of Shp2-D61G or Shp2-A462T in zebrafish
embryos induced cell movement defects, resulting in elongated
embryos at bud stage (10 hpf ) (Fig. 7A-C). Treatment with 0.25 μM
CI-1040 at 4.5 hpf for 1 h rescued development at 10.5 hpf of
embryos expressing Shp2-D61G and Shp2-A462T (Fig. 7D-F).
Quantification of the epiboly defects confirmed the rescue of the
embryos expressing NS and LS Shp2 variants (Fig. 7G). In addition,
we confirmed that elevated pERK levels in embryos expressing
Shp2-D61G and Shp2-A462T were reduced by CI-1040-mediated
inhibition of MEK at 10 hpf (Fig. 7H).
Next, the effect of CI-1040 treatment was assessed on asymmetry of
the heart in embryos expressing Shp2-D61G and Shp2-A462T.
Treatment with 0.25 μM CI-1040 at 4.5 hpf for 1 h rescued leftward
displacement of the heart at 24 hpf of Shp2-D61G and Shp2-A462Texpressing embryos (Fig. 7I,J). However, treatment of these embryos
for 1 h with CI-1040 at the 10-somite stage (14 hpf) did not restore
normal heart displacement in embryos expressing NS and LS Shp2
variants (Fig. 7K). Treatment with 0.25 μM CI-1040 at 4.5 hpf for 1 h
largely rescued randomization of the left-right asymmetry markers
and of the liver (supplementary material Fig. S5). These results
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DEVELOPMENT
sonic hedgehog marker (Krauss et al., 1993) indicated that
expression of Shp2-D61G and Shp2-A462T altered left-right
patterning without disrupting the midline, which provides a
barrier between the left and the right side (supplementary material
Fig. S4). These results show that L/R patterning in the LPM is
impaired in embryos expressing NS and LS Shp2 variants, which
may lead to randomization of heart and gut laterality.
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Development (2014) 141, 1961-1970 doi:10.1242/dev.106310
Fig. 6. Impaired ciliogenesis and cilia function in Kupffer’s vesicle in embryos expressing Shp2-D61G and Shp2-A462T. (A-C) Immunohistochemistry
was carried out using anti-acetylated tubulin on WT-Shp2-, Shp2-D61G- or Shp2-A462T-expressing embryos that were fixed at the 10-somite stage.
Representative confocal images are depicted here. Scale bars: 50 μm. Insets show close-ups of cilia. (D-F) Quantification of the cilia number, cilia length and KV
area in WT-Shp2-, Shp2-D61G- and Shp2-A462T-expressing embryos that were treated with 0.5% DMSO (control) or 0.25 μM CI-1040 at 4.5 hpf for 1 h and fixed
at the 10-somite stage (n=10 for each condition). Averages are depicted with error bars indicating s.e.m. Statistical significance was determined using
Student’s t-test: ***P<0.001; **P<0.01. (G-L) Tracks of fluorescent beads injected in KV. (G-L) Maximum projections of fluorescent bead movements injected
in the KV of WT-Shp2-, Shp2-D61G- or Shp2-A462T-expressing embryo that were treated with 0.5% DMSO or 0.25 μM CI-1040 at 4.5 hpf for 1 h and fixed
at the 10-somite stage. (M) Average flow velocity of beads in KV of embryos expressing WT-Shp2, Shp2-D61G and Shp2-A462T without (−) or with (+)
CI-1040 treatment. The number of beads/number of embryos that were analyzed is indicated. Averages are depicted with error bars indicating s.e.m.
Statistical significance was determined using Student’s t-test: ***P<0.001.
1966
counterclockwise rotation of the fluorescent beads in Shp2-D61Gand Shp2-A462T-expressing embryos to levels that were similar to
those in WT-Shp2-expressing embryos (Fig. 6J-M). Taken together,
these results suggest that the cardiac defects in zebrafish embryos
expressing NS and LS Shp2 variants are due to hyperactivate
MAPK-induced laterality defects originating from impaired cilia
function in KV.
DISCUSSION
In this paper, we used zebrafish to investigate defects in cardiac
development in response to expression of NS and LS variants of
Shp2. We found that NS- and LS-Shp2-expressing embryos showed
impaired leftward heart displacement. Our results are consistent
with a report that Noonan-associated mutations induce a cardiac
looping defect in Xenopus (Langdon et al., 2012). Here, we provide
insight into the underlying mechanism. The cardiac defects that we
observed in zebrafish embryos are associated with L/R laterality
defects and with defects in ciliogenesis. Expression of NS and LS
variants of Shp2 in zebrafish embryos enhanced MAPK activation.
The laterality defects were rescued by early treatment with CI-1040,
DEVELOPMENT
indicate that inhibition of MEK at early stages rescues L/R asymmetry
in embryos expressing NS and LS Shp2-variants.
Finally, the effect of CI-1040 treatment on ciliogenesis and cilia
function in KV was assessed. The cilia number was not rescued
upon treatment with 0.25 μM CI-1040 at 4.5 hpf for 1 h. In fact,
the cilia number in CI-1040-treated WT-Shp2-expressing embryos
was significantly reduced to similar levels as in CI-1040-treated
embryos expressing NS or LS variants of Shp2 (Fig. 6D). Yet,
CI-1040 treatment rescued cilia length in embryos expressing
Shp2 variants (3.6±0.1 μm in Shp2-D61G- and 3.8±0.1 μm in Shp2A462T-expressing embryos, compared with 3.2±0.1 μm in
untreated embryos) (Fig. 6E). Cilia length in WT-Shp2-expressing
embryos was reduced somewhat in response to CI-1040 treatment
(from 4.2±0.1 μm to 3.7±0.1 μm; Fig. 6E) and hence, CI-1040
treatment abolished the difference in cilia length between embryos
expressing WT-Shp2 and Shp2 variants. Moreover, CI-1040
treatment rescued KV area (3736±373 μm2 in Shp2-D61G- and
3055±294 μm2 in Shp2-A462T-expressing embryos, compared
with 3986±398 μm2 in WT-Shp2-expressing embryos; Fig. 6F).
Importantly, treatment with 0.25 μM CI-1040 at 4.5 hpf rescued the
RESEARCH ARTICLE
Development (2014) 141, 1961-1970 doi:10.1242/dev.106310
a MEK inhibitor, indicating that the defects in heart asymmetry were
caused by enhanced MAPK signaling before gastrulation.
Here, we focused on the cardiac defects in embryos expressing NS
and LS Shp2 variants. The heart defects only became evident during
cardiac tube formation, the first asymmetric process that occurs in the
zebrafish heart (Bakkers, 2011). Normally, the cardiac tube is
displaced to the left, but in embryos expressing NS and LS Shp2
variants, heart displacement was randomized (Fig. 3). Cell movement
analyses revealed a delay in cell movements, coupled with reduced
cardiac tube rotation, resulting in morphologically abnormal hearts at
28 hpf (Fig. 4). Cell tracking studies showed that loss of Nodal or
BMP signaling leads to similar defects (de Campos-Baptista et al.,
2008; Smith et al., 2008). Nodal mutant embryos show reduced speed
and directional movement of the cardiomyocytes, resulting in
disruption of the leftward morphogenesis and cardiac cone rotation.
Asymmetric spaw expression in the LPM is responsible for the correct
displacement of the heart tube to the left (Long et al., 2003; de
Campos-Baptista et al., 2008). In line with these findings, we found
that expression of spaw was randomized in embryos expressing NS
and LS Shp2 variants, concomitant with its downstream gene target
lft2. As a consequence, left/right asymmetry was lost and leftward
displacement of the heart in embryos expressing Shp2-D61G and
Shp2-A462T was compromised. In addition, randomized fabp
expression suggested that not only asymmetry of the heart, but also
of the liver was impaired.
Left-right asymmetry is conserved across species and defects
in left-right asymmetry are collectively called laterality disease
(Mercola and Levin, 2001). The phenotype we observed in
zebrafish embryos expressing NS and LS Shp2 variants was
characterized by randomization of left/right asymmetry. It is
noteworthy that laterality disease is frequently associated with
congenital heart defects (CHDs) (Ramsdell, 2005). Impaired leftright patterning can affect cardiac morphogenesis, resulting in
defective septation and/or double outlet right ventricle (DORV)
(Ramsdell, 2005). Cardiac defects associated with laterality diseases
are also reported in Shp2-D61G knock-in mice. A proportion of the
heterozygous Shp2-D61G mice displays septal defects and DORV
(Araki et al., 2004). However, laterality defects are not the only
cause of septation defects or DORV. Other laterality defects were
not reported in NS or LS Shp2 knock-in mice, and laterality defects
have not been commonly reported in humans with NS and LS.
Hence, it remains to be determined whether laterality defects have a
role in human NS and LS.
In zebrafish expressing NS and LS variants of Shp2, we found
that the underlying mechanism for randomization of L/R
asymmetry is likely impaired cilia function in the KV. In
particular, cilia length and KV area were associated with Shp2
variant-induced impaired fluid flow in KV and subsequent loss of
asymmetry (Fig. 6). It is well established that L/R asymmetry is
mediated by cilia that generate fluid flow within the KV, resulting
in expression of spaw in the left side of the LPM (Nonaka et al.,
1998; Essner et al., 2005). Laser-mediated ablation of KV
randomizes expression of spaw and lft2 (Essner et al., 2005).
Moreover, shorter cilia result in perturbed intravesicular fluid flow,
leading to loss of asymmetry in the heart, brain and viscera
(Ferrante et al., 2009). KV formation is completed by the 6-somite
1967
DEVELOPMENT
Fig. 7. Early treatment with the MEK-inhibitor CI-1040
rescued leftward heart displacement and cilia length in
embryos expressing NS and LS Shp2 variants. Embryos
were injected with mRNA encoding WT-Shp2 (Shp2),
Shp2-D61G and Shp2-A462T at the one-cell stage.
(A-F) Representative images of bud stage embryos are depicted.
The shape of the NS- and LS-Shp2-expressing embryos is
restored by treatment with 0.25 μM CI-1040 at 4.5 hpf for 1 h.
(G) The ratio between the length of the major and minor axes
was determined as quantitative measure of the epiboly defects
in NS- and LS-Shp2-expressing embryos. Averages are
depicted with error bars indicating s.e.m. Statistical significance
was determined using Student’s t-test (**P<0.01; ***P<0.001).
(H) Embryos were treated with MEK inhibitor CI-1040 (0.25 μM)
for 1 h at 4.5 hpf or mock-treated with DMSO and lysed at
10 hpf. Immunoblots of the zebrafish lysates were stained using
antibodies specific for pErk and Erk. The experiment was
repeated three times and representative blots are depicted here.
(I-K) Embryos were mock treated with DMSO (I) or with CI-1040
for 1 h at 4.5 hpf (J) or at 10 hpf (K). Heart displacement was
determined in embryos following in situ hybridization with a
myl7-specific probe as in Fig. 4. Histograms represent the
percentage of embryos with heart displacement to the left,
middle or right. The total number of embryos (n) is indicated.
RESEARCH ARTICLE
1968
Whereas MAPK activation may be elevated by both NS and LS
variants of Shp2, elevated PI3K/AKT signaling is exclusively
associated with LS. Hyperactivation of AKT signaling in response
to LS-Shp2 is responsible for HCM in mouse hearts (Ishida et al.,
2011; Marin et al., 2011; Schramm et al., 2012). We also observed
elevated pAkt levels in response to LS-Shp2 in zebrafish embryos
(supplementary material Fig. S3), but we did not observe HCM in
these embryos. The cardiac defects in embryos expressing NS and
LS Shp2 variants were indistinguishable. Moreover, these heart
defects were rescued by early treatment with the MEK inhibitor to a
similar extent, indicating a causal role for elevated MAPK activation
in the observed cardiac defects, independently of Akt signaling.
In summary, our data provide new insights into the role of
pathogenic Shp2 variants in cardiac development. Expression of NS
and LS Shp2 variants led to similar cardiac defects in zebrafish that
were associated with impaired L/R asymmetry, resulting from
impaired cilia function in KV. Treatment with a MEK inhibitor
prior to gastrulation rescued cilia function, L/R asymmetry and heart
displacement, suggesting a causal relation with enhanced MAPK
signaling. No studies to date have examined the effect of NS and LS
Shp2 variants on L/R asymmetry. Our paper opens a new prospective
in understanding how pathogenic SHP2 and downstream MAPK
activation relates to cardiac development through its effects on L/R
asymmetry.
MATERIALS AND METHODS
Fish line
Zebrafish were maintained and the embryos were staged as previously
described (Kimmel et al., 1995). The tg(myl7:GFP) line has been previously
described (Chocron et al., 2007). All procedures involving experimental
animals were approved by the local animal experiments committee and
performed in compliance with local animal welfare laws, guidelines and
policies, according to national and European law.
Constructs, RNA and injections
The eGFP-peptide 2A-Shp2 construct was derived by PCR. 50 capped sense
mRNAs were synthesized using the mMessage mMachine kit (Ambion) and
linearized plasmid DNA. mRNA injections were performed at the one-cell
stage as described previously (Hyatt and Ekker, 1999) using optimized amounts
of mRNA (D61G=150 pg, A465T=50 pg, T73I=80 pg G465A=100 pg,
WT-Shp2=150 pg).
Immunoblotting
Zebrafish embryos (10 hpf ) were lysed in buffer containing 50 mM Tris
(pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM sodium orthovanadate, 1%
Nonidet P-40, 0.1% sodium deoxycholate, protease inhibitor mixture
(Complete Mini, Roche Diagnostics) and vanadate. Samples were run on
SDS-PAGE gel (10%), transferred to PVDF membrane and stained with
Coomassie Blue to verify equal loading. The blots were probed
with antibodies specific for SHP2, Actin, pERK, ERK, pAKT, AKT (all
Cell Signaling) and GFP (Torrey Pines). Detection was carried out using
enhanced chemiluminescence (Thermo Scientific kit).
In situ hybridization and immunofluorescence microscopy
In situ hybridization was carried out essentially as described (Thisse et al.,
1993) using probes specific for, myl7 (Yelon et al., 1999), vmhc (Yelon,
2001), amhc (Smith et al., 2009), has2 (Bakkers et al., 2004), lefty2
(Bisgrove et al., 1999), southpaw (Long et al., 2003), fabp and shh (Krauss
et al., 1993).
For zebrafish immunohistochemistry, embryos were fixed in 4% DENT’s
solution (20% dimethylsulfoxide/80% methanol) at 4°C, and subsequently
blocked for 1 h in PBS containing 5% lamb serum, 1% BSA, 1% DMSO
and 0.1% Triton-X. Embryos were incubated in mouse anti-acetylated
tubulin (1:500, Sigma) for 3 h at room temperature. After extensive washing,
embryos were incubated in goat anti-mouse Alexa Fluor 488. Embryos were
DEVELOPMENT
stage (12 hpf ) (Oteiza et al., 2008, 2010) and the generation of the
KV depends on the dorsal forerunner cells (DFCs) that are induced
during the blastula period (Alexander et al., 1999). Defects in the
specification, clustering or organization of DFCs induce defective
KV organogenesis and/or ciliogenesis (Essner et al., 2005; Oteiza
et al., 2010). Therefore, it will be interesting to investigate the role
of the NS and LS Shp2 variants in DFC biology.
Shp2 is a well-known downstream factor in FGFR signaling (Neel
and Tonks, 1997). It has been reported that FGFR1 signaling
regulates cilia length in zebrafish. Morpholino (MO)-mediated
knockdown of FGFR1 in zebrafish reduces cilia length in KV and
perturbs directional fluid flow, thus impairing L/R patterning of the
embryo (Neugebauer et al., 2009). The observation that knockdown
of FGFR1, a positive regulator of MAPK signaling, has similar
effects to mutant Shp2-induced hyperactivation of MAPK appears to
be contradictive. However, it is not uncommon that attenuation
and activation of a signaling pathway have the same effect on
developmental processes. FGFR1 knockdown, on the one hand, and
Shp2 variant-mediated enhanced MAPK activation, on the other,
have similar effects on cilia function in KV and both impair L/R
asymmetry. Our results predict that ectopic activation of MAPK
signaling, e.g. overexpression of (activated) MAPK, around
gastrulation would also induce laterality defects.
Defective ciliogenesis was observed in the KV of NS and
LS-Shp2-expressing embryos. It remains to be determined
whether ciliogenesis in other organs is also affected in these
embryos. It is noteworthy that deafness is associated with LS in
humans. Deafness is typically associated with defective cilia in the
inner ear and future work should focus on ciliogenesis in organs
other than KV in NS and LS Shp2-variant-expressing zebrafish
embryos.
The cardiac defects that were induced by expression of NS and
LS Shp2 variants in zebrafish embryos were indistinguishable.
MAPK activation was elevated at bud stage in response to
expression of both Shp2-D61G (NS) as well as Shp2-A462T (LS)
(Fig. 7H), which might explain why we observed similar
developmental defects at early stages. It is well established that
NS-Shp2 mutations enhance MAPK activation, but LS-Shp2
often does not activate MAPK signaling. Nevertheless, it is not
unprecedented that LS mutations induce defects that are associated
with activation of MAPK signaling. Expression of LS-Shp2
mutants in Drosophila results in ectopic wing veins and a rough
eye phenotype, characteristics of increased Erk activation. This
gain-of-function phenotype is similar to that of NS-Shp2
transgenic flies (Oishi et al., 2006, 2009). Moreover, a recent
study shows that induced pluripotent stem cells (iPSCs) derived
from the fibroblasts of individuals with LS display higher basal
pERK levels compared with those of control iPSCs (CarvajalVergara et al., 2010). How the NS and LS variants of Shp2 would
both activate MAPK signaling remains to be determined. Whereas
catalytic activity of Shp2 is enhanced by the NS mutations, it is
reduced by the LS mutations, indicating that MAPK activation
is not directly affected by alterations in Shp2 catalytic activity.
Rather, the NS and LS Shp2-variant-induced defects may result
from a phosphatase-independent function of Shp2. Alternatively,
as suggested in a recent report, the LS variants of SHP2 retain
some catalytic activity and mediate gain-of-function phenotypes,
because they have an increased propensity for the open
conformation. Thus, the LS variants bind upstream activators
preferentially and stay in complex with their scaffolding adapters
longer, thus prolonging specific substrate turnover (Yu et al.,
2013), which in turn may lead to MAPK activation.
Development (2014) 141, 1961-1970 doi:10.1242/dev.106310
RESEARCH ARTICLE
Pharmacological inhibition of MAPK signaling
To test the prevention of the NS/LS Shp2 mRNA phenotype, embryos
injected with NS/LS Shp2 mRNA were incubated with 0.25 μM CI-1040 at
4.5 hpf or 10 hpf for 1 h at 28.5°C in the dark.
Time-lapse imaging and analysis
Embryos at the 22-somite stage were dechorionated and mounted in glassbottom six-well plates using 0.25% agarose in E3 embryo medium
containing 16 mg/ml 3-amino benzoic acid ethylester to block contractile
movements. Confocal imaging was performed using a spinning disc
confocal laser scanning microscope with 20× magnification, acquiring
stacks every 5 min. Embryos were kept at 28.5°C during recordings. ImageJ
software (http://rsb.info.nih.gov/ij/) was used to generate time-lapse movies
and for cell counting. Cell tracking was carried out using Volocity software
(Improvision) followed by manual inspection of individual tracks generating
quantification of total speed (track length/time). Rotation was calculated by
measuring the angle with the LR axis of four imaginary lines connecting
four individual cells at the start and the end of the time-lapse (200 min). All
statistical analyses were performed in Excel (Microsoft) using the two-tailed
Student’s t-test.
KV fluid flow and cilia motility
Fluorescent beads (Polysciences) were injected into KV to visualize flow as
described previously (Essner et al., 2005). Bead flow was imaged at the 8- to
10-somite stage (SS) on a Leica AF7000 microscope (Leica Microsystems
GmbH, Wetzlar, Germany) using a 63× water dipping objective and an
Hamamatsu C9300-221 high-speed CCD camera (Hamamatsu Photonics,
Hamamatsu City, Japan) at 80 fps to record a 10 s movie. Movies were
generated using ImageJ software. Bead tracking was carried out using
Volocity software (Improvision). For each embryo, four or five beads were
tracked for a minimum of 50 frames of the movie and the average bead
velocity was calculated.
High-speed imaging and analysis
Embryos (2 dpf ) were mounted in 0.25% agarose prepared in E3 medium
embryonic medium with 16 mg/ml 3-amino benzoic acid ethylester.
Embryonic hearts were imaged with a Hamamatsu C9300-221 highspeed CCD camera (Hamamatsu Photonics, Hamamatsu City, Japan) at
150 fps mounted on a Leica AF7000 microscope (Leica Microsystems
GmbH, Wetzlar, Germany) in a controlled temperature chamber (28.5°C)
using Hokawo 2.1 imaging software (Hamamatsu Photonics GmbH,
Herrsching am Ammersee, Germany). Image analysis was carried out
with ImageJ (http://rsbweb.nih.gov/ij/). Statistical analysis and drawing
of the box-whisker plot were carried out in Excel 2007 (Microsoft,
Redmond, WA, USA).
Statistics
Cilia measurements were analyzed using the two-tailed Student’s t-test.
Averages for controls and experimental were compared within each clutch of
embryos. Results were considered significant when P<0.05 and results are
expressed as mean±s.e.m. (*P<0.05, **P<0.01, ***P<0.001).
Competing interests
The authors declare no competing financial interests.
Author contributions
J.d.H. conceived the project; M.B. and J.d.H. designed the approach with help from
F.T., E.N. and J.B.; M.B., J.P.O., F.T. and E.N. performed experiments; M.B. and
J.d.H. prepared the manuscript, which was edited by J.P.O., F.T., E.N. and J.B. prior
to submission.
Funding
This work was funded, in part, by a grant from the Research Council for Earth and
Life Sciences [ALW 819.02.021] with financial aid from the Netherlands
Organisation for Scientific Research (NWO) (to J.d.H.). Deposited in PMC for
immediate release.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.106310/-/DC1
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